CN109423517A - Purposes of the excretion body in diagnosing tumor, treatment and prognosis evaluation - Google Patents
Purposes of the excretion body in diagnosing tumor, treatment and prognosis evaluation Download PDFInfo
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Abstract
Provided herein is purposes of the excretion body in diagnosing tumor, treatment and prognosis evaluation.Specifically, the purposes of lung cancer for prognosis is used to diagnose and/or treat lung cancer and/or assessed provided herein is excretion body.A kind of composition and/or kit is also provided herein comprising excretion body analysis agent and/or regulator.For the present invention for the key molecule mechanism of research invasion of lung cancer, transfer and regulation, improving lung cancer therapy effect and assessment patients with lung cancer prognosis has important realistic meaning.
Description
Technical field
This application involves tumor diagnosis and therapy fields, specifically, including inhibiting this application involves excretion body regulator
Application of the agent in the diagnosing and treating of tumour such as lung cancer.
Background technique
Excretion body (Exosomes) is the nanoscale capsule that the diameter that Trams etc. had found at first in 1980 is 40~130nm
Bubble structure plays important wherein including a large amount of protein, nucleic acid and lipid etc. in intercellular substance information exchanging process
Effect[11,12].Compared with normal cell, tumour cell discharges more excretion bodies, and a series of eggs included in it
White matter and nucleic acid component play crucial work in adjusting tumor microenvironment, the invasion for promoting tumour and orientation transfer process
With[13,14].Furthermore the prognosis mala of the high-level excretion body and tumor patient that generate some researches show that tumour cell has significant
Correlation[14]。
Lung cancer, which has become, threatens one of maximum malignant tumour to human health, and disease incidence is in obviously to increase in majority state
Trend, having more than 1,000,000 people dies of lung cancer every year in the whole world, wherein about 85% is non-small cell lung cancer (Non-Small Cell
Lung Cancer, NSCLC), in China, the morbidity and mortality of lung cancer are in the first place of all malignant tumours[1,2].At present
The treatment of NSCLC includes operation, radiotherapy, chemotherapy etc., but operative treatment is still the currently the only possible means for curing lung cancer, only not
The patient of sufficient half has an opportunity to receive radical surgery treatment, and postoperative 5 years survival rates are only 30-60%.Although chemotherapy can improve
The survival rate of patient NSCLC, however 5 annual survival rates are only 15%；Generally speaking, receive adjuvant chemotherapy of patients mid-term patient and
Receive the patients with terminal of chemicotherapy, clinical prognosis is not fully up to expectations[3,4].Clinical data shows the invasion of NSCLC and transfer is shadow
The key factor of patient's curative effect and existence is rung, but since the transfer regulatory mechanism of NSCLC is complex, clinically still lacked effectively
Invasion of lung cancer transfer correlation predictive mark and possible targeted inhibition means.
Local infiltration and far-end transfer are the most important features of malignant tumour, and this is also the main of lung cancer causing death
Reason[5].Existing numerous studies discovery, compared with normal cell, it is micro- to adjust tumour that tumour cell discharges more active factors
Environment[6], to escape the killing of immune system[7], and enhance tumor cell invasion transfer ability；Regulatory factor in microenvironment simultaneously
It can influence the survival and proliferation of far-end transfer tumour cell[8,9].The microenvironment of entity tumor includes different cytokines and various
The function of extracellular matrix etc., performance is not quite similar, and its constituent has differences in different entity tumors[10].In recent years
Research shows that tumour cell is mainly by a variety of regulatory factors of the outside active secretion of different size of vesica, and wide with form of vesicles
It is general to be present in tumor infiltative lymphocyte and body fluid, it is established with the microenvironment of tumour closely related[9]。
Bibliography
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Cancer J Clin.2016；66 (2): 115-32
2.Siegel RL, Miller KD, Jemal A, et al.Cancer statistics, 2015.CA Cancer
J Clin.2015；65 (1): 5-29
3.Torre LA, Siegel RL, Jemal A.Lung Cancer Statistics.Adv Exp Med
Biol.2016；893:1-19
4.Johnson DH, Schiller JH, Bunn PA Jr.Recent clinical advances in lung
cancer management.J Clin Oncol.2014；32 (10): 973-82
5.Inamura K, Ishikawa Y.Lung cancer progression and metastasis from
the prognostic point of view.Clin Exp Metastasis.2010；27 (6): 389-97
6.Yuana Y, Sturk A, Nieuwland R.Extracellular vesicles in physiological
and pathological conditions.Blood Rev.2013；27 (1): 31-9
7.Robbins PD, Morelli AE.Regulation of immune responses by
extracellular vesicles.Nat Rev Immunol.2014；14 (3): 195-208
8.Whiteside TL.The tumor microenvironment and its role in promoting
tumor growth.Oncogene.2008；27 (45): 5904-12.
9.Tkach M, Th é ry C.Communication by Extracellular Vesicles:Where We
Are and Where We Need to Go.Cell.2016；164 (6): 1226-32
10.Tang H, Qiao J, Fu YX.Immunotherapy and tumor
microenvironment.Cancer Lett.2016；370 (1): 85-90
11.Trams EG, Lauter CJ, Salem N Jr, et al.Exfoliation of membrane ecto-
enzymes in the form of micro-vesicles.Biochim Biophys Acta.1981；645 (1): 63-70
12.Penfornis P, Vallabhaneni KC, Whitt J, et al.Extracellular vesicles
As carriers of microRNA, proteins and lipids in tumor microenvironment.Int J
Cancer.2016；138 (1): 14-21
13.Becker A, Thakur BK, Weiss JM, et al.Extracellular Vesicles in
Cancer:Cell-to-Cell Mediators of Metastasis.Cancer Cell.2016；30 (6): 836-848
14.Lobb RJ, Lima LG,A.Exosomes:Key mediators of metastasis and
Pre-metastatic nicheformation.Semin Cell Dev Biol.2017.doi:10.1016/
j.semcdb.2017.01.004.[Epub ahead of print]
15.Wang X, Liu Y, Liu X, et al.MiR-124inhibits cell proliferation,
migration and invasion by directly targeting SOX9in lung adenocarcinoma.Oncol
Rep.2016；35 (5): 3115-21
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MicroRNA, miR-34and miR-124Family, Methylation on Prognosis of Non-Small-Cell
Lung Cancer.Clin Lung Cancer.2017；18 (1): e13-e20
17.Cai QQ, Dong YW, Wang R, et al.MiR-124inhibits the migration and
invasion of human hepatocellular carcinoma cells by suppressing integrinαV
expression.Sci Rep.2017；7:40733
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prognosis in pancreatic ductal ademoeareinoma patients.Br J Biomed Sci.2016；
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suppressor in breast cancer by targeting CBL.BMC Cancer.2016；16 (1): 826.
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21.Bobrie A, Krumeich S, Reyal F, et al.Rab27a supports exosome-
dependent and-independent mechanisms that modify the tumor microenvironment
and can promote tumor progression.Cameer Res.2012；72 (19): 4920-30.
22.Ostrowski M, Carmo NB, Krumeich S, et al.Rab27a and Rab27b control
different steps of the exosome secretion pathway.Nat Cell Biol.2010；12 (1): 19-
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Summary of the invention
Inventor, which studies, finds that the secretion of normal pulmonary epithelial cells excretion body is substantially less than NSCLC tumor cell line (p <
0.001), and the excretion body of tumor cell secretion has facilitation to the invasion transfer of tumour cell in vitro；Serum levels
Research finds that excretion body content is significantly higher than normal population (p < 0.001) in NSCLC patients serum, and divides with the clinic of patient
Phase has positive correlation.
In some embodiments, the present invention relates to excretion bodies to prepare for diagnosing and/or treating lung cancer and/or assessment
The composition of lung cancer for prognosis and/or the purposes in kit.
In some embodiments, the composition of the invention and/or kit include excretion body analysis agent and/or tune
Agent is saved, including analysis agent tolerant and/or vesicle surface albumen in such as excretion body and/or excretion body regulation of secretion agent, including
Such as detecting the tolerant reagent as existing for albumen or nucleic acid in excretion body and/or excretion body, including for example with patients with lung cancer
In the detection agent and/or analysis agent of relevant molecule are especially secreted in pulmonary metastasis patient to excretion body.
In some embodiments, the lung cancer of the invention includes Small Cell Lung Cancer and non-small cell lung cancer.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA and/or detection and/or the reagent for adjusting one or more of protein.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA includes miR-4485, miR-124, miR-326, miR-6803, miR-4433, miR-
4286, miR-199, miR-4306, miR-3120, miR-33 and/or miR-1246, miR-144, miR-148a, miR-200b,
MiR-150, miR-342, miR-29a, miR-1304, miR-10a, miR-7g.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA include miR-33, miR-124, miR-126, miR-203, miR-128b, miR-221,
MiR-7a, miR-199 and/or miR-137, miR-372, miR-182, miR-221, miR-130, miR-15b, miR-145.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA includes miR-33, miR-124, miR-199.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of greater protein matter, the protein include excretion body regulation of secretion albumen, including Rab family GTP enzyme, such as Rab, especially
It is Rab27a and Rab27b, sphingomyelinase 2 (SMS2), soluble n-ethylmaleimide sensitive factor attachment complex
(SNAREs), such as syntaxin-4 and SNAP-23 and VAMP-2 and VAMP-8.
In some embodiments, the composition of the invention and/or kit include detecting and/or adjusting miR-
124 reagent and/or detection and/or the reagent for adjusting Rab27a.
In some embodiments, the present invention relates to a kind of composition and/or kits comprising one or more of above
The reagent of description.
Detailed description of the invention
Fig. 1: low-level miR-124 passes through the release for improving the expression regulation excretion body of Rab27a kinases in tumour cell,
The final invasion transfer and prognosis for influencing NSCLC.
Fig. 2:
A: the secretion level (0.74 × 10 of normal pulmonary epithelial cells HBEpiC excretion body9/ mL) it is substantially less than NSCLC tumour
Cell strain SPC-A-1 and NCI-H1299 (1.62 × 109/mL、2.11×109/mL；P < 0.001)；
B: excretion body content (4.04 × 10 in serum levels research discovery NSCLC patients serum11/ mL) it is significantly higher than normally
Crowd (1.19 × 1010/mL；P < 0.001), and there is positive correlation with the progress of disease and the clinical stages of patient；
C: the NSCLC patient shifted, the excretion body content in serum are significantly higher than the patient not shifted；
D: cell scratch experiment is research shows that the NCI-H1299 excretion body of various concentration moves tumour cell SPC-A-1
Shifting ability has direct facilitation, and there are dose-effect relationships for effect.
Fig. 3: normal control tissue, NSCLC do not shift patient and shift the table of miRNAs in the primary lung neoplasm tissue of patient
Up to difference, clustering obtains the significant miRNA molecule of preceding 20 kinds of differential expressions.
Fig. 4:
After A:miR-124 and control plasmid transfect, the size distribution trend of the excretion vesica of NSCLC cell NCI-H1299:
The excretion body of 40-130nm proportion in cell Secretory vesicles is larger in control tumor cell；After miR-124 is overexpressed, carefully
The ratio of excretion body significantly reduces in intracrine vesica；
B:miR-124 is overexpressed, and significantly suppresses the release of excretion body；
C: the intracellular miR-124 of NSCLC is overexpressed reduction excretion body level to fluorescence co-focusing as the result is shown.
The reduction of Fig. 5: miR-124 level significantly improves the secretion level of excretion body.
Fig. 6: the miR-124 influence to intracellular excretion body secretion Regulated Proteins expression.
Specific embodiment
Inventor has found that patients with lung cancer especially NSCLC patient is related to the secretion of excretion body, and it was found that excretion
The secretion of body is related to the invasion of tumour and transfer.It therefore, can be by detecting and/or monitoring and/or adjust in tumor patient
The secretion of excretion body come diagnose and/or treat lung cancer and/or assessment lung cancer for prognosis.
As described above, in some embodiments, the present invention relates to excretion body in preparation for diagnosing and/or treating lung cancer
And/or the purposes of assessment lung cancer for prognosis.It in some embodiments, can be by detecting and/or monitoring and/or adjusting tumor patient
Lung cancer and/or assessment lung cancer for prognosis are diagnosed and/or treated such as the excretion body secretion of the tumour cell of patients with lung cancer.
In some embodiments, the composition of the invention and/or kit include excretion body analysis agent and/or tune
Agent is saved, including analysis agent tolerant and/or vesicle surface albumen in such as excretion body and/or excretion body regulation of secretion agent, including
Such as detecting the tolerant reagent as existing for albumen or nucleic acid in excretion body and/or excretion body, including for example with patients with lung cancer
In the detection agent and/or analysis agent of relevant molecule are especially secreted in pulmonary metastasis patient to excretion body.The analysis of excretion body
It is known in the art, such as can also passes through antibody test excretion body surface markers by being centrifugated excretion body.Example
As in some embodiments, excretion body analysis agent of the present invention includes reagent existing for determining excretion body, including for example
Detect the antibody of excretion body surface markers, the especially antibody of the surface markers of detection patients with lung cancer excretion body specificity.In addition,
Surface plasma resonance (SPR) has also been used to quantify and detect excretion body and its surface markers.The excretion body analysis agent
It further include determining such as albumen or core tolerant and/or relevant to excretion body in tumor patient in detection excretion body and/or excretion body
Reagent existing for acid (including DNA and/or RNA, such as miRNA).Detection albumen and/or nucleic acid existing for method be this field
Know, including is for example detected using specific antibody and/or complementary series.This field has also been provided for purifying excretion
The kit of body and analysis RNA molecule, such as Qiagen exoRNeasy Serum/Plasma Maxi Kit (Exosome
Diagnostics, Inc).In some embodiments, method of the invention and/or product can be in the samples from tumor patient
It is carried out on product such as tumour cell and/or blood sample such as serum.
Herein, the excretion body analysis agent and/or regulator can be used for diagnosing and/or assess the pre- of patients with lung cancer
Afterwards.In some embodiments, the excretion body analysis agent and/or regulator can be also used for the treatment of lung cancer.For example,
It was found that miRNA includes miR-33, the secretion of excretion body in the adjustable tumor patient such as miR-124, miR-199.In some realities
It applies in scheme, the present invention relates to the secretions that excretion body in tumor patient is reduced by miRNA, to reduce tumor invasiveness.?
In some embodiments, the present invention relates to include miR-33, the expression of miR-124, miR-199, suppression for example, by improving miRNA
Excretion body regulation of secretion albumen processed includes Rab27a, inhibits the excretion body of tumour cell to secrete, to treat tumour.Therefore, exist
It include miR-33, the expression and inhibition excretion of miR-124, miR-199 the present invention relates to miRNA is improved in some embodiments
Body regulation of secretion albumen includes the combination of the reagent of Rab27a.In addition, in some embodiments, the present invention relates to raisings
MiRNA and/or the reagent and the excretion body regulation of secretion protein expression of raising and/or reduction different approaches for reducing miRNA expression
Reagent combination.Such as the present invention can provide the reagent and adjust other than Rab27a for improving that miRNA includes miR-124 expression
Excretion body regulation of secretion agent combination.The invention further relates to the combinations for the reagent for detecting above-mentioned miRNA and/or albumen, are used for
Diagnose and/or treat lung cancer and/or assessment lung cancer for prognosis.In these areas, it is anticipated that by the detections of different approaches and/or
The combination of regulative mode can be improved the sensitivity and/or specificity of diagnosing tumor and/or treatment.
In some embodiments, the lung cancer of the invention includes Small Cell Lung Cancer and non-small cell lung cancer.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA and/or detection and/or the reagent for adjusting one or more of protein.Detection nucleic acid and/or protein are deposited
Reagent be known in the art, and can readily prepare.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA includes miR-4485, miR-124, miR-326, miR-6803, miR-4433, miR-
4286, miR-199, miR-4306, miR-3120, miR-33 and/or miR-1246, miR-144, miR-148a, miR-200b,
MiR-150, miR-342, miR-29a, miR-1304, miR-10a, miR-7g.It has have been surprisingly discovered that in metastatic lung
In cancer, miR-4485, miR-124, miR-326, miR-6803, miR-4433, miR-4286, miR-199, miR-4306,
The expression of miR-3120, miR-33 reduce and/or miR-1246, miR-144, miR-148a, miR-200b, miR-150, miR-
The expression of 342, miR-29a, miR-1304, miR-10a, miR-7g, which are increased, (does not shift with normal control and patients with lung cancer phase
Than).Therefore, in some embodiments, the present invention can be by detecting miR-4485, miR-124, miR-326, miR-
The expression of 6803, miR-4433, miR-4286, miR-199, miR-4306, miR-3120, miR-33 reduce and/or miR-
1246, miR-144, miR-148a, miR-200b, miR-150, miR-342, miR-29a, miR-1304, miR-10a, miR-
The expression of 7g increases diagnosing and/or assesses the prognosis situation of lung cancer.In some embodiments, miR-4485, miR-
The expression of 124, miR-326, miR-6803, miR-4433, miR-4286, miR-199, miR-4306, miR-3120, miR-33
Reduction and/or miR-1246, miR-144, miR-148a, miR-200b, miR-150, miR-342, miR-29a, miR-1304,
Instruction pulmonary metastasis is increased in the expression of miR-10a, miR-7g and/or the prognosis of patient is poor.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA include miR-33, miR-124, miR-126, miR-203, miR-128b, miR-221,
MiR-7a, miR-199 and/or miR-137, miR-372, miR-182, miR-221, miR-130, miR-15b, miR-145.?
Through have beeing surprisingly discovered that in patients with lung cancer, miR-33, miR-124, miR-126, miR-203, miR-128b, miR-221,
The expression of miR-7a, miR-199 reduce and/or miR-137, miR-372, miR-182, miR-221, miR-130, miR-15b,
The expression of miR-145 increases (compared with the expression of normal pulmonary epithelial cells).Therefore, in some embodiments, miR-33,
The expression of miR-124, miR-126, miR-203, miR-128b, miR-221, miR-7a, miR-199 reduce and/or miR-
The expression increase of 137, miR-372, miR-182, miR-221, miR-130, miR-15b, miR-145 can be used for diagnosis or auxiliary
Help diagnosing.
In some embodiments, of the present invention any to diagnose and/or treat and/or prognosis evaluation mode be made
It any is diagnosed and/or treated and/or prognosis evaluation mode is combined for supplementary mode with other.In some embodiments,
It is diagnosed and/or treated and/or prognosis evaluation mode by incorporated in the present invention, improves and diagnose and/or treat and/or prognosis is commented
The sensitivity and/or specificity estimated and/or effect.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of more miRNA, the miRNA includes miR-33, miR-124, miR-199.It has have been surprisingly discovered that in lung cancer
In patient, miRNA includes miR-33, and the expression of miR-124, miR-199 reduce.In some embodiments, miRNA includes
The expression of miR-33, miR-124, miR-199 change the secretion of adjustable excretion body.
In some embodiments, the composition of the invention and/or kit include detection and/or adjust it is a kind of or
The reagent of greater protein matter, the protein include excretion body regulation of secretion albumen, including Rab family GTP enzyme, such as Rab, especially
It is Rab27a and Rab27b, sphingomyelinase 2 (SMS2), soluble n-ethylmaleimide sensitive factor attachment complex
(SNAREs), such as syntaxin-4 and SNAP-23 and VAMP-2 and VAMP-8.
In some embodiments, the composition of the invention and/or kit include detecting and/or adjusting miR-
124 reagent and/or detection and/or the reagent for adjusting Rab27a.In some embodiments, the composition of the invention
And/or kit include detection and/or adjust miR-124 reagent and/or detection and/or adjust other than adjusting Rab27a
Excretion body regulation of secretion albumen, including Rab family GTP enzyme such as Rab27b, sphingomyelinase 2 (SMS2), soluble N- ethyl
Maleimide sensitive factor attachment complex (SNAREs), such as syntaxin-4 and SNAP-23 and VAMP-2 and
VAMP-8.It is contemplated that the detection and/or adjusting of the regulative mode by combination different approaches, can be improved the spirit of diagnosing tumor
Quick property and/or specificity, can also improve the therapeutic effect of tumour.
In some embodiments, the present invention relates to a kind of composition and/or kits comprising one or more of above
The reagent of description.For example, in some embodiments, the present invention provide detection agent and/or therapeutic agent and/or diagnosticum and/or
Compositions related and/or kit comprising determine excretion body and/or correlation point in tumor cells specimens and/or blood sample
Reagent existing for son (as excretion body surface reaches adjusting molecule in excretion body label, tumour-specific content substance markers, tumour cell).
In some embodiments comprising adjust the reagent that the excretion body surface of tumor patient reaches, such as reduce the examination that excretion body surface reaches
Agent, the reagent can be such as miRNA molecule.
Further describe some embodiments of the invention below in conjunction with specific embodiment, the embodiment is not construed as pair
Concrete restriction of the invention.
Research method:
1) the coherent detection analysis of excretion body
The separation and Extraction of excretion body:Tumour cell in vitro culture 72 hours, serum free medium is replaced, it is small to continue culture 48
When, supernatant is collected by centrifugation in 1500g, after 10000g centrifugation removes cell fragment, Nanosight can be used directly and carry out excretion body point
The detection of the amount of secreting and excretion body size distribution.For serum specimen, directly progress centrifugation step, subsequent step is the same as cell conditioned medium mark
This.
The quantitative counting of excretion body:It is directly detected using excretion body of the Nanosight to said extracted, obtains excretion
The related data of body quantity and size distribution.
2) culture and transfection of tumour cell
NSCLC cell line used in experiment is NCI-H1299 and SPC-A-1；Control cell is on immortalized bronchial
Chrotoplast HBEpiC, using the RMPI-1640 culture medium for containing 10% fetal calf serum, in 37 DEG C, 5%CO2Under conditions of cultivate；
Cell purchase is saved from consonance tumour library, this room, is detected and is verified through STR.
It is overexpressed the transfection of plasmid:The luciferase reporting plasmid that expression plasmid uses save this seminar early period
PcDNA3.1 is constructed；For cell inoculation in 6 orifice plates, cell quantity is 3 × 105A/hole, every hole add 2ml containing 10%FBS
Culture medium is placed in 37 DEG C of incubators and cultivates 18-24h, and transfection experiment can be carried out when its degrees of fusion reaches 70%-80%.
Cell transfecting is carried out using lipo2000, transfection process is operated according to specification.
Inhibit the transfection of miRNA:Respectively by Lipofetaamine RNAiMAX and the 9ul miRNA (10umol/L) of 9ul
It is dissolved in the Opi-MEM solution of 150ul, Lipofetaamine RNAiMAX dilution is added dropwise in miRNA dilution
In, it is incubated at room temperature 5min, takes 250ul mixed liquor to be added in 6 orifice plates, is put into 37 DEG C of cell incubator.
3) miRNA influences the research approach of tumor cell invasion transfer
The detection of cell migration power:Cell transfecting miR-124 analogies and after negative control 24 hours, with 1%FBS's
It is 2 × 10 that RMPI-1640 culture medium, which adjusts cell concentration,8/ L, takes 200ul that upper chamber is added, and lower room is added 600ul and contains 10%FBS
RMPI-1640 culture medium, 37 DEG C of incubation 18h remove the cell Transwell, and PBS cleans the residual cells on cell upper layer, 4%
Paraformaldehyde room temperature fixes 15min, 0.1% violet staining 40min, and migrating cell number, every film are counted under 200 power microscopes
The transmission cell number for counting upper, middle and lower or so the 5 different visuals field, calculates average value, every group sets 3 multiple holes, with the phase of migrating cell
Logarithm amount indicates the variation of the transfer ability of tumour cell.
The detection of cell invasion ability:Matrigel basilar memebrane cell will be coated in cell invasion kit to prevent setting room temperature
It is spare that 37 DEG C of incubation 1-2h of 300ul serum-free RMPI-1640 culture medium are added in balance.Cell transfecting miR-124 analogies and yin
Property control 24 hours after, be 5 × 10 with the RMPI-1640 culture medium of 1%FBS adjustment cell concentration8/ L takes in 300ul addition
The RMPI-1640 culture medium of 500ul FN containing 10%FBS and 5mg/L, 37 DEG C of incubation 48h is added in room, lower room, and cell is fixed and contaminated
The same Cell migration assay of color step, the variation of the invasive ability of tumour cell is indicated with the relative populations of invasion cell.
(CAM) is tested in the invasion transfer ability of body chick chorioallantoic membrane detection tumour:Obtain the egg of fertilization and by egg
37 DEG C in MultiQuip incubator, it is incubated under 60% humidity.Aseptically at the 3rd day of developing chick embryo in shell
On make an osculum.Window is resealed with adhesive tape, and egg is put back in incubator until the 11st day of chick embryo development.The 11st
It, by NCI-H1299 cell suspending liquid (9 × 105) with matrigel (8.9mg/mL, the BD of growth factor reduction
Biosciences it) mixes, total volume is 30 μ L.Control group is the miR-124 (20 μm of ol/mL) and NCI-H1299 of nano-encapsulated
Cell and matrigel are mixed together.Matrigel mixture is placed at the top of CAM, reseal and returns to incubator 72 hours is (every
N=6 chicken embryo of a cell line).Embryo and surrounding CAM matrigel are harvested, and fixes 24 hours with 4% paraformaldehyde, is embedded in
In paraffin.Serial section hematoxylin and eosin dyeing.Using NanoZoomer (Hamamatsu Photonics K.K.) into
Row digital scan.
4) research approach of miRNA molecule Targeted-control target gene
Using TargetScan, the informatics softwares such as miRanda and Microcosm Targets analyze miRNA and excretion
Body secretion related gene and possible action site, selective analysis secrete directly related Rab family with excretion body.
Firstly, directly verifying the relationship of target protein Yu miR-124 expression using WB immunoblot assay；
Secondly, using the method for fluorescent co-location, it was demonstrated that direct regulation and control of the miRNA molecule to downstream molecules；And it utilizes
Luciferase system identifies the 3 '-UTR regions in conjunction with miRNA molecule；
Finally, the identification using Dual-luciferase reportor systerm to target molecule: extracting the mankind from healthy human blood cell
Genomic DNA, designs the PCR primer containing xho1 and Not1 double digestion taste, and amplifying target genes 3 ' hold non-coding region (3 '
UTR)；Psi-CHECK2- [Target gene] -3 '-UTR-wt wild type recombinant vector is constructed, while designing miR-124 and mesh
Gene junction mutant nucleotide sequence synthesize psi-CHECK2- [Target gene] -3 '-UTR-mut saltant type recombinant vector.It answers
Transfected wild-type and saltant type recombinant vector are distinguished into 293T cell with Lipofectamine 2000, each carrier setting is empty
White control group, miR-124 inhibition group and inhibition negative control group.It is said according to promega company Dual-Luciferase detection kit
Bright book operation, detects the fluorescence intensity of cell.
5) research approach of the upstream miR-124 regulatory molecule
Gene association analysis:Research is found out using existing information in Cancer Genome Atlas (TCGA) database
Data set in NSCLC research, explores in NSCLC and with miRNA-124 expresses consistent gene, selects wherein that consistent degree is most
Five kinds of high genes carry out subsequent analysis；
Genetic enrichment analysis:By carrying out gene probe enrichment analysis (GSEA) to NSCLC tissue samples, above-mentioned five are found out
Most possible upstream modulin molecule in a associated gene；Research is analyzed using GSEA 2.2.1 editions software.According to
The expression of miRNA-124 is divided into high expression group and low expression group.It is obtained in the MsigDB database of the website this research and utilization GSEA
Obtain data set；Then, enrichment analysis is carried out by the method for default weights enrichment statistics, setting random combine number is 1000 times.
6) detection scheme of cell and protein level and miRNA level in tissue
Cell and albumen in flesh tissue sample and miRNA use Western blot and RT-PCR method；Paraffin group
The albumen for knitting sample slice and miRNA detection are using IHC and ISH detection method.Specific experiment step operates to specifications, and WB is real
It tests and Bio-rad companies scan instrument analysis result is shone and passed through using ECL.IHC is developed the color using DAB and is worked using pathological image
It stands scanning, the cell that as a result positive criteria is in each visual field 25% shows weakly positive to strong positive.
7) animal shift experiment is studied
The experiment of vein metastasis model:By 8 week old male NOD/SCID mouse tail vein injections 1 × 106/ 200 μ l fluoresceins
Enzyme gene and miR-124 gene are total to plasmid transfection NCI-H1299 cell and control NCI-H1299 cell.It injects preceding and injection
Luciferase expression cell is monitored using IVIS Lumina XRMS series III imaging system (PerkinElmer) weekly afterwards.It is small
Mouse presses 300mg/kg weight intraperitoneal injection 100mL D- fluorescein (Biosynth；Cas#115144-35-9), then isoflurane
It is detected after gas anesthesia.
The experiment of lung cancer original position metastasis model:The male NOD/SCID mouse of 8 week old, using puncture needle by 1.5 × 105/200
The above-mentioned miR-124 of μ l is overexpressed NCI-H1299 and control NCI-H1299 cell mass is inoculated into mouse lung, and presses above-mentioned inspection
Survey method is imaged.After animal is put to death, exteriorize in vitro biodiversity resources.
Subcutaneous metastasis model experiment:The male NOD/SCID mouse of 8 week old is subcutaneously inoculated with 5 × 10 respectively6It is a above-mentioned
MiR-124 high expresses NCI-H1299 cell or control NCI-H1299 tumour cell, reaches 0.7-0.9cm to tumor volume3Afterwards,
It is imaged according to above-mentioned detection method.After animal is put to death, exteriorize in vitro biodiversity resources.
Metastases Inhibition test:MiR-124 is given in targeting to be influenced in the experiment of invasion of lung cancer transfer, dynamic to above-mentioned three groups
Three treatment groups are respectively set, respectively abdominal cavity in object experiment, the miR-124 molecule of tail vein and intratumor injection nano-encapsulated, weekly
Injection is twice.Every 3 days measurement tumours simultaneously record.
8) statistical method: miR-124 concentration is indicated with mean+SD.Between expression and clinical and pathological data
Correlation is examined by Mann-Whitney U；Measurement data is examined with t or variance analysis.Other qualitative datas are examined using card side
It tests and is analyzed.All data analyses are completed using SPSS13.0 statistical package in experiment, and use 6.0 software of Prism
Mapping.Statistics uses two-sided test, and P < 0.05 thinks with statistical significance.
(1) relationship of NSCLC cell excretion body and invasion transfer.
Inventor studies the secretion level (0.74 × 10 for finding normal pulmonary epithelial cells HBEpiC excretion body9/ mL) it is significant
Lower than NSCLC tumor cell line SPC-A-1 and NCI-H1299 (1.62 × 109/mL、2.11×109/mL；P < 0.001) (figure
2A)；Excretion body content (4.04 × 10 in serum levels research discovery NSCLC patients serum11/ mL) it is significantly higher than normal population
(1.19×1010/mL；P < 0.001), and there is positive correlation (Fig. 2 B) with the progress of disease and the clinical stages of patient, especially
Interestingly, the NSCLC patient shifted, the excretion body content in serum are significantly higher than the patient's (figure not shifted
2C).Result of study shows the invasion transfer of excretion body and NSCLC, and there are significant correlations.It is thin to tumour in order to verify excretion body
Born of the same parents invade the direct regulation and control effect of transfer, and inventor has carried out cell scratch experiment in vitro, research shows that various concentration
NCI-H1299 excretion body has direct facilitation to the transfer ability of tumour cell SPC-A-1, and there are dosage effects for effect
It should be related to (Fig. 2 D), research prompt, the excretion body of tumour cell release, which may shift the invasion of NSCLC, has significant regulation
Effect.
(2) the miRNA molecule Differential expression analysis of excretion body release is influenced in NSCLC tumour cell.
Inventor compares normal control tissue, NSCLC first and does not shift patient and the transfer primary lung neoplasm tissue of patient
The differential expression of middle miRNAs obtains the significant miRNA molecule (Fig. 3) of preceding 20 kinds of differential expressions by clustering inventor.
Inventor compares lung cancer cell line NCI-H1299, SPC-A-1 and normal bronchial epithelial cell line HBEpiC simultaneously, passes through
The method of miRNA sequencing, obtains the miRNA molecule queue (table 1) of differential expression；In conjunction with this two research in obtain with
There are associated miRNA molecules for NSCLC patient's transfer and excretion body secretion difference, and it is relevant to further reduce influence lung cancer
MiRNA molecule list；And subsequent verifying and analysis are carried out to the most significant miRNA of differential expression.
Fig. 2 shows that the source different number NCI-H1299 excretion body is made with the promotion to the transfer of SPC-A-1 cell invasion
With.
The excretion body secretory volume of A.NSCLC cell line NCI-H1299 and SPC-A-1 are significantly higher than immortalization pulmonary epithelial cells
HBEpiC；Excretion body content is significantly higher than no tubercle normal control population in B.NSCLC patients serum, and sends out with disease
The relationship of consistency is presented in exhibition；Excretion body content is significantly higher than the patient not shifted in the serum of C.NSCLC transfer patient.D. outer
Secreting body has significant facilitation to tumor cell migration ability.
Fig. 3 shows that normal control and NSCLC do not shift and shift the survey of Patients with Lung in situ tumor tissue miRNAs molecule
Sequence analysis, in pulmonary metastasis, miR-4485, miR-124, miR-326, miR-6803, miR-4433, miR-4286,
The expression of miR-199, miR-4306, miR-3120, miR-33 reduce and/or miR-1246, miR-144, miR-148a, miR-
The expression of 200b, miR-150, miR-342, miR-29a, miR-1304, miR-10a, miR-7g are increased (with normal control and not
Transfer patients with lung cancer is compared).
The differential expression of table 1.NSCLC cell NCI-H1299, SPC-A-1 and miRNA in normal pulmonary epithelial cells
(3) NSCLC intracellular miR-124, miR-199 and miR-33 is overexpressed the level for significantly reducing excretion body.
Inventor has carried out the early screening marker screening study of non-small cell lung cancer, the wherein research in the direction miRNAs
The result shows that a variety of miRNAs and NSCLC clinical progress and transfer are significant related in tumour cell.Inventor has collected two kinds
NSCLC representativeness cell strain (NCI-H1299 and SPC-A-1), to NSCLC cell strain and normal immortal bronchial epithelial cell
(HBEpiC) total miRNAs is sequenced in excretion body, filters out 15 groups and the significant relevant differential expression of excretion body secretion level
MiRNAs molecule.By after result early period and sequencing result combined analysis, 3 kinds of inventor's Primary Study are secreted and lung with excretion body
The significant relevant miRNA molecule (miR-124, miR-199 and miR-33) of metastasis of cancer.
PcDNA3.1-miR-124 expression plasmid is constructed, NCI-H1299 is transfected by gene, is demonstrated in cellular level
High-level miR-124 can inhibit the release of excretion body, as a result as shown in Figure 4.The excretion body of 40-130nm in control tumor cell
Proportion is larger in cell Secretory vesicles；After miR-124 is overexpressed, the ratio of excretion body significantly drops in cell Secretory vesicles
Low (Fig. 4 A).Quantitative analysis results show that excretion body secretion level reduces by 75% or more after miR-124 is overexpressed.Fluorescence copolymerization
The above results have been proved in coke experiment, and after intracellular miR-124 is overexpressed, excretion body level is significantly reduced, and prompt miR-124 may
It is the key molecule of NSCLC cell regulation excretion body.(Fig. 4 C).MiR-199 and miR-33, which is overexpressed, can get similar results.
Fig. 4 shows that NSCLC cell miR-124 is overexpressed the influence to excretion body level.
After A.miR-124 and control plasmid transfect, the size distribution trend of the excretion vesica of NSCLC cell NCI-H1299；
B.miR-124 is overexpressed, and significantly suppresses the release of excretion body.C. the fluorescence co-focusing intracellular miR-124 of NSCLC as the result is shown
Being overexpressed, which reduces miR-124 level reduction in the horizontal pulmonary epithelial cells of excretion body, significantly improves the secretion level of excretion body.
HBEpiC, which is immortalized, by pulmonary branches tracheae backward transfects CD63-GFP/miR-124 antisense nucleic acid coexpression into the cell
After plasmid, the 4th hour replacement culture medium, continuous monitoring is it is experimentally confirmed that miR-124 strikes drop and intracellular excretion body level
Increase significant correlation, and excretion body is gradually secreted into extracellularly, the reduction of miR-124 level is as a result prompted to be remarkably improved excretion body
Secretion level.(Fig. 5).
Fig. 5 shows that the reduction of miR-124 level significantly improves the secretion level of excretion body.
Research regulates and controls excretion by being overexpressed miR-124, desk study miR-124 in NSCLC cell strain NCI-H1299
Body secretes the change of related protein expression level.MiR-124 is significantly suppressed into the cell Western blot as the result is shown
Rab27a protein expression (Fig. 6), result of study prompt the effect of miR-124 regulation excretion body may be with intracellular Rab27a albumen
Molecule is directly related.
Fig. 6 shows influence of the miR-124 to intracellular excretion body secretion Regulated Proteins expression.
It is analyzed by TargetScan online tool, the mechanism that Rab27a is expressed in desk study miR-124 regulating cell.
Using TargetScan online tool, inventor analyzes possibility site of the miR-124 in conjunction with Rab27a,
As a result, it has been found that 3 ' UTR region of Rab27a gene has 3 possible binding sites (table 2).For these three sites, inventor's difference
Mutant nucleotide sequence is devised, rite-directed mutagenesis is carried out to 3 ' UTR sequences, gene order, which has designed, at present finishes, and is ready for next
The verifying of step.
Table 2.miR-124 and the possible binding site of Rab27a
Research shows that in patient tumors cell low-level miR-124 may by improve Rab27a kinases expression regulation outside
The release of body is secreted, the final invasion transfer and prognosis (Fig. 1) for influencing NSCLC.The secretion of tumour cell excretion body is had studied herein to change
The molecular regulation process of change, it is expected that illustrating NSCLC cell influences the specific molecular mechanism of invasion transfer by excretion body.It is above-mentioned to grind
Studying carefully result will be helpful to illustrate the molecular mechanism that the release of NSCLC tumour cell excretion body increases, and expand invasion of lung cancer route of metastasis
Ken, and new provide important clue and new view for lung cancer metastasis target spot and transfer prognosis evaluation index to develop
Angle.
Claims (10)
1. excretion body is in preparation for diagnosing and/or treating in lung cancer and/or the composition and/or kit of assessing lung cancer for prognosis
Purposes.
2. purposes described in claim 1, wherein the composition and/or kit include excretion body analysis agent and/or adjusting
Agent, including analysis agent tolerant and/or vesicle surface albumen in such as excretion body and/or excretion body regulation of secretion agent, including example
As for detecting the tolerant reagent as existing for albumen or nucleic acid in excretion body and/or excretion body, including for example and in patients with lung cancer
The detection agent and/or analysis agent of relevant molecule are especially secreted in pulmonary metastasis patient to excretion body.
3. purposes of any of claims 1 or 2, wherein the lung cancer includes non-small cell lung cancer.
4. the purposes in claim any one of 1-3, wherein the composition and/or kit include detecting and/or adjusting one
The reagent of kind or more and/or detection and/or the reagent for adjusting one or more of protein.
5. the purposes in claim any one of 1-4, wherein the composition and/or kit include detecting and/or adjusting one
Kind or more reagent, the miRNA includes miR-4485, miR-124, miR-326, miR-6803, miR-4433,
MiR-4286, miR-199, miR-4306, miR-3120, miR-33 and/or miR-1246, miR-144, miR-148a, miR-
200b, miR-150, miR-342, miR-29a, miR-1304, miR-10a, miR-7g.
6. the purposes in claim any one of 1-5, wherein the composition and/or kit include detecting and/or adjusting one
The reagent of kind or more, the miRNA includes miR-33, miR-124, miR-126, miR-203, miR-128b, miR-
221, miR-7a, miR-199 and/or miR-137, miR-372, miR-182, miR-221, miR-130, miR-15b, miR-
145。
7. the purposes in claim any one of 1-6, wherein the composition and/or kit include detecting and/or adjusting one
The reagent of kind or more, the miRNA includes miR-33, miR-124, miR-199.
8. the purposes in claim any one of 1-6, wherein the composition and/or kit include detecting and/or adjusting one
Kind or more reagent, the protein includes excretion body regulation of secretion albumen, including Rab family GTP enzyme, such as Rab,
Especially Rab27a and Rab27b, sphingomyelinase 2 (SMS2), soluble n-ethylmaleimide sensitive factor attachment are multiple
Fit (SNAREs), such as syntaxin-4 and SNAP-23 and VAMP-2 and VAMP-8.
9. the purposes in claim any one of 1-7, wherein the composition and/or kit include detection and/or adjust
The reagent of miR-124 and/or detection and/or the reagent for adjusting the excretion body regulation of secretion albumen in addition to Rab27a.
10. a kind of composition and/or kit comprising reagent defined in any one of claim 1-9.
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